The union of sperm and egg at fertilization is an essential step in reproduction. In mammals, including human, sperm must first bind to the egg zona pellucida and complete the acrosome reaction before fusing with the egg. The zona pellucida initiates acrosome reactions by driving calcium entry into sperm through TRPC channels. However, the mechanisms by which the zona pellucida regulates the conductance through these sperm ion channels are poorly understood and represent a major gap in our knowledge. Based on preliminary and published data, a working hypothesis is developed that the zona pellucida drives the synthesis of phosphatidylinositol-4, 5-bisphosphate (PIP2) and that this lipid regulates the function of two members of the TRPC channel family by two distinct mechanisms. The aims of this proposal are to: 1) determine the mechanism by which the zona pellucida regulates PIP2 synthesis and metabolism; 2) determine the mechanisms by which that PIP2 controls calcium conductance through TRPC channels; and 3) determine how sperm capacitation and seminal plasma components contribute to TRPC channel function and to fertilization. These studies are relevant in two regards. First, they may advance our understanding of the basic biology of fertilization. Second, there may be therapeutic consequences of these studies both with regard to the treatment of human infertility and for the design of novel contraceptives. In particular, the effects of PIP2 metabolites that are produced in sperm in response to zona pellucida stimulation are directly relevant to the control of the human acrosome reaction.